SUMO Modification Regulates BLM and RAD51 Interaction at Damaged Replication Forks
et al. (2009) SUMO Modification Regulates BLM and RAD51 Interaction at Damaged Replication
Forks. PLoS Biol 7(12): e1000252. doi:10.1371/journal.pbio.1000252
SUMO Modification Regulates BLM and RAD51 Interaction at Damaged Replication Forks
Karen J. Ouyang 0 1
Leslie L. Woo 0 1
Jianmei Zhu 0 1
Dezheng Huo 0 1
Michael J. Matunis 0 1
Nathan A. Ellis 0 1
James E. Haber, Brandeis University, United States of America
0 Current address: Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts , United States of America
1 1 Committee on Genetics, Genomics, and Systems Biology, University of Chicago , Chicago , Illinois, United States of America, 2 Department of Biochemistry and Molecular Biology, University of Chicago , Chicago , Illinois, United States of America, 3 Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology, Johns Hopkins University , Baltimore , Maryland, United States of America, 4 Department of Health Studies, University of Chicago , Chicago , Illinois, United States of America, 5 Department of Medicine, University of Chicago , Chicago, Illinois , United States of America
The gene mutated in Bloom's syndrome, BLM, is important in the repair of damaged replication forks, and it has both proand anti-recombinogenic roles in homologous recombination (HR). At damaged forks, BLM interacts with RAD51 recombinase, the essential enzyme in HR that catalyzes homology-dependent strand invasion. We have previously shown that defects in BLM modification by the small ubiquitin-related modifier (SUMO) cause increased c-H2AX foci. Because the increased c-H2AX could result from defective repair of spontaneous DNA damage, we hypothesized that SUMO modification regulates BLM's function in HR repair at damaged forks. To test this hypothesis, we treated cells that stably expressed a normal BLM (BLM+) or a SUMO-mutant BLM (SM-BLM) with hydroxyurea (HU) and examined the effects of stalled replication forks on RAD51 and its DNA repair functions. HU treatment generated excess c-H2AX in SM-BLM compared to BLM+ cells, consistent with a defect in replication-fork repair. SM-BLM cells accumulated increased numbers of DNA breaks and were hypersensitive to DNA damage. Importantly, HU treatment failed to induce sister-chromatid exchanges in SM-BLM cells compared to BLM+ cells, indicating a specific defect in HR repair and suggesting that RAD51 function could be compromised. Consistent with this hypothesis, RAD51 localization to HU-induced repair foci was impaired in SM-BLM cells. These data suggested that RAD51 might interact noncovalently with SUMO. We found that in vitro RAD51 interacts noncovalently with SUMO and that it interacts more efficiently with SUMO-modified BLM compared to unmodified BLM. These data suggest that SUMOylation controls the switch between BLM's pro- and anti-recombinogenic roles in HR. In the absence of BLM SUMOylation, BLM perturbs RAD51 localization at damaged replication forks and inhibits fork repair by HR. Conversely, BLM SUMOylation relieves its inhibitory effects on HR, and it promotes RAD51 function.
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Funding: This work was supported by the Blooms Syndrome Foundation (http://www.milogladsteinfoundation.org/), The Cancer Research Foundation (http://
www.cancerresearchfdn.org/), the Department of Medicine at the University of Chicago (http://www.uchicago.edu/), the Digestive Disease Research Core Center
(P30 DK42086), the Molecular and Cellular Biology training grant (GM007183 to KJO), University of Chicago Cancer Research Center, and the Biological Sciences
Division. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of this manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: BS, Blooms syndrome; DSB, double-strand break; HR, homologous recombination; PFGE, pulsed-field gel electrophoresis; PML-NB,
promyelocytic leukemia nuclear body; SCE, sister-chromatid exchange; ssDNA, single-stranded DNA.
. These authors contributed equally to this work.
Homologous recombination (HR) is a high-fidelity DNA repair
mechanism that functions to rejoin double-strand breaks (DSBs)
and restart broken replication forks. A major outcome of the repair
of replication fork damage by HR is the generation of
sisterchromatid exchanges (SCEs), which result from resolution of
Holliday junctions during HR repair [1,2]. Predictably, a large
number of agents that cause DNA damage increase the
frequencies of SCEs [35]. Blooms syndrome (BS) is the only
clinical entity in which increased levels of SCE are a prominent
cellular feature [6]. It is an autosomal recessive disorder, which is
characterized by proportional dwarfism, photosensitivity,
immunodeficiency, hypogonadism, and predisposition to a wide range of
different types of cancer [7]. BS is caused by biallelic null
mutations of the BLM gene [8]. The BLM gene encodes a DNA
helicase of (...truncated)